Subject: Big Problems with Novagenīs HisTag Protein Purification
From: "Thorsten Schmidt"
Date: 9 Nov 1996 16:05:31 GMT
Dear Sir or Madam!
I have big Problems with Novagen`s HisTag Protein Purification-Kit.
I use the pET30-Vector which allows translation of my target protein
containing
6 His-residues. The target protein expression is inhibited during cell
growth and
induced by IPTG addition.
The 6 Histidines are used for protein-purification:
The Histidines bind to a "His bind resin" carrying Ni ions.
One should get 50-100 mg total protein after cell lysis and -after
purification- 20 mg target protein from one 100 ml culture.
But which differences between theory and my experience!!!
I followed strikly Novagenīs purification protocol but the problems begin
with the
total protein. Instead of the predicted 50-100 mg, I get only 2 (!).
But there might be no fault possible: One just harvests the cells by
centrifugation, resuspends the cells in a buffer (containing 5 mM imidazol,
0,5 M NaCl and 20 mM Tris-HCl pH 7,9), sonicates and then removes debris by
ultra centrifugation (39.000 g).
Changes in the protocol (addition of Lysozyme, 0,1 % Triton) increases the
total protein amount only to 30 mg.
This leads to my first question:
What can I also do to increase the total protein yield?
May Lysozyme or Triton lead to problems with the HisTag purification?
The second problem is the colomn chromatographie:
According to the protocol I expect a flow rate of 25 ml/h.
But the real flow rate is just 10 ml/h. Filtration of the cell lysate or
new packing of
the column did not help.
Although SDS-PAGE analysis showed that the target protein is expressed, I
got no target protein (0,04 mg instead of 20 mg!).
Have you made experiences with this purification kit?
Why is the total protein concentration so low?
How can I increase the columnīs flow rate?
Why is it impossible to purify my target protein?
Can you help me?
Thank you for your answer!
yours sincerely
Thorsten Schmidt
Subject: CANCER, GENERAL QUESTIONS
From: ehaynes@fas.harvard.edu (Elizabeth Haynes)
Date: 11 Nov 1996 00:25:16 GMT
I'm researching cancer incidence and treatment in the US, France and
Germany. Anyone out there who has any experience or expertise with cancer
-- patients, doctors, nurses, dentists, etc. -- and can help me answer
some of the questions in my research, your time is greatly appreciated.
Basically, I am focusing on the following areas, primarily as they relate
to cancers of the head and neck, liver and esophagus:
How are medical diagnoses made? By whom?
Who treats these various cancers and where (hospitals, cancer
centers, etc.)? How significant a role does one's insurance plan play in
the diagnosis/referral/treatment process? Any other factors (geography,
etc.)?
How are the above processes different in France and Germany?
If you have ANY experience or expertise in the area of cancer, PLEASE post
me a message back or write to me at:
ehaynes@fas.harvard.edu
Thanks so much!
emh.
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Elizabeth M. Haynes *
ehaynes@fas.harvard.edu *
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